Autoreactive T cells that are capable of inducing disease exist in normal adult animals, but are maintained in a dormant or inactive state due to the suppressive functions of regulatory T cells. We have demonstrated that the regulatory T cells can be easily identified in normal lymphoid tissues by co-expression of CD4 and the interleukin-2 receptor alpha chain (CD25). Transfer of CD4+ CD25- T cells to immunoincompetent mice results in the development of autoimmune disease that can be prevented by co-transfer of CD4+CD25+ cells. Our recent studies have focused on defining the mechanism of action of the CD4+CD25+ in vitro and in vivo: (1) We have analyzed the effects of polyclonal regulatory T cells (Treg) on the development of autoimmune gastritis (AIG). Treg inhibited the development of disease, but failed to inhibit the migration of the effector cells into the gastric lymph node (gLN) or into the stomach. Notably, Treg did not inhibit the expansion of autoreactive T cells in the gLN. The primary effect of the Treg appeared to be inhibition of the differentiation of the autoantigen-specific T cells to Th1 effector cells as reflected by a decrease in antigen-stimulated IFN-gamma production and reduction in Tbet expression. (2) We have studied the capacity of dendritic cells (DC) to activate naturally occurring CD4+CD25+ Treg as well as the ability of CD4+CD25+ T cells to suppress the DC-mediated activation of CD4+CD25- T cells. Mature bone marrow derived-DC (BMDC), but not splenic DC, were able to induce the proliferation of CD4+CD25+ T cells in the presence of a polyclonal stimulus and in the absence of exogenous IL-2. The DC-induced proliferative response of the CD4+CD25+ T cells was partially dependent on IL-2 produced by a small number of contaminating CD25+ effector cells. We measured IL-2 production in co-cultures of CD4+CD25+ and CD4+CD25- T cells using the IL-2 secretion assay. Surprisingly, CD4+CD25+ T cells markedly suppressed IL-2 secretion by the CD4+CD25- T cells without inhibiting their proliferation. Collectively, these results suggest that antigen presentation by DC can induce the expansion of CD4+CD25+ T cells, while simultaneously activating their ability to suppress cytokine secretion by effector T cells. (3) Although the suppressive capacity of Treg on T cell activation has been well documented. Little is know concerning the effects of Treg on other cell types that are involved in immune responses. By co-culturing pre-activated CD4+CD25+ T cells with B cells in the presence of polyclonal B cell activators, we found that B cell proliferation was significantly suppressed. The suppression of B cell proliferation was due to increased cell death caused by the CD4+CD25+ T cells in a cell-contact dependent manner. The induction of B cell death is not mediated by Fas-Fas ligand pathway, but surprisingly depends on the up-regulation of perforin and granzymes in the CD4+CD25+ T cells. Our results demonstrated that CD4+CD25+ T cells can act directly on B cells and suggest that the efficacy of CD4+CD25+ T cells in preventing autoimmunity can be explained, at least in part, by the direct regulation of B cell function. (4) Treg are thought to have T cell receptors (TCRs) with intermediate to high affinity for self, below the threshold for negative selection, but sufficient to induce a differentiation signal that results in their unique phenotype. Treg freshly isolated from lymph nodes have a high level of constitutive TCR zeta-chain phosphorylation that is consistent with their enhanced reactivity with self. The signal delivered to Treg that results in zeta-chain phosphorylation initiates or sustains a program where the regulatory T cells can be activated to mediate T suppressor function in vitro in the presence of IL-2/IL-4 without further stimulation via their TCR. This result raises the possibility that Treg are continuously activated by recognition of self on DC and by cytokines produced by effectors as part of normal T cell homeostasis. In an inflammatory environment in which cytokines are produced in abundance, Treg may represent a highly efficient immunologic rapid response force that exists in a partially primed, activated state that is capable of responding to signals received from activated effector cells. (5) We find that a portion of polyclonal CD4+CD25+ T cells divides extensively when transferred into normal recipients. This division is dependent upon recipient MHC class II expression, and the dividing cells express FoxP3. To study the fate of natural regulatory T cells during an immune response, we followed a tracer population of Treg co-transferred with ovalbumin-specific TCR-Tg CD4+CD25- T cells. Following immunization with OVA peptide, the dividing, but not the quiescent Treg subset is preferentially recruited into both the draining lymph nodes and the inflamed tissue, despite their lack of specificty for the antigen. This recruitment requires effector T cell activation, but is independent of effector-derived IL-2. When isolated from unimmunized mice, this Treg subset is capable of directly mediating antigen non-specific suppression without additional TCR stimulation, implying they have been activated in vivo. These data strongly suggest that a subpopulation of Treg activated by recognition of self-peptide/MHC class II molecules are preferentially recruited into inflammatory sites following effector T cell stimulation. By suppressing early events following T cell activation, this subset may play a vital role in decreasing the magnitude of the initial inflammatory response. (6) It is unclear how the minor population of Treg cells is capable of exerting its powerful suppressor effects. To explore the possibility that Treg cells inhibit immune responses by converting naive T cells to the suppressor phenotype, we co-cultured CD4+CD25+ T cells with naive CD4+CD25- from TCR transgenic mice in the presence of anti-CD3 and IL-4 to promote cell viability. When the responder cells were labeled with CFSE, two distinct cell populations could be recovered from the co-culture. We found that cells in the CFSE high fraction were non-responsive to re-stimulation with anti-CD3, or exogenous IL-2, and could not up-regulate IL-2 mRNA or CD25 expression upon TCR stimulation. These CFSE high cells were capable of suppressing the response of fresh CD4+CD25- T cells and CD8+ T cells. In contrast, the responders cells that divided were anergic, responded to IL-2, and were only marginally suppressive. The induced suppressor cells did not express the transcription factor, Foxp3, which is expressed by Treg. We conclude that Treg may induce cells from the naive T cell pool to become suppressors and this may be one of the mechanisms responsible for infectious tolerance. Taken together, our studies provde a strong background for undertanding the pleiotropic effects of Treg on immune responses in autoimmunity, tumor immunity, and infectious diseases. Manipulation of Treg function should represent a novel adjunct to the therapy of several diseases.